Tryptophan hydroxylase (EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)) in rat brainstem extracts is activated 2 to 2.5-fold by ATP and Mg ions in the presence of subsaturating concentrations of the cofactor, 6-methyltetrahydropterin (6MPH4). The activation of tryptophan hydroxylase under these conditions results from a reduction in the apparent Km for 6MPH4 from 0.21 mM to 0.09 mM. The activation required Mg ions and ATP but is not dependent on either cAMP or cGMP. The effect of ATP and Mg ions on enzyme activity was enhanced by micron M concentrations of Ca ions and totally blocked by EGTA. These data suggest that tryptophan hydroxylase can be activated by a cyclic nucleotide independent protein kinase which requires low calcium concentrations for the expression of its activity.